Method of producing a fermentable sugar

Abstract

Provided herein is a transgenic bacteria engineered to accumulate carbohydrates, for example disaccharides. Also provided is a photobioreactor for cultivating photosynthetic microorganisms comprising a non-gelatinous, solid cultivation support suitable for providing nutrients and moisture to photosynthetic microorganisms and a physical barrier covering at least a portion of the surface of the cultivation support. Devices for the large scale and continuous cultivation of photosynthetic microorganisms incorporating photobioreactors and methods of use are disclosed. Also disclosed are methods of producing fermentable sugar from photosynthetic microorganisms using a photobioreactor of the invention.

What is claimed is: 
     
       1. A method of producing a fermentable sugar using a photobioreactor, the method comprising:
 (A) inoculating a photobioreactor with a transgenic cyanobacteria engineered to accumulate at least one fermentable sugar selected from the group consisting of sucrose and trehalose, wherein the transgenic cyanobacteria comprises an artificial DNA construct comprising
 (i) a promoter functional in the transgenic cyanobacteria; 
 (ii) a nucleotide sequence encoding
 (a) a first polypeptide comprising SEQ ID NO: 2 or a sequence 95% identical thereto having sucrose phosphate synthase and sucrose phosphate phosphatase (ASF) activity; or 
 (b) a second polypeptide and a third polypeptide, 
 
 
 wherein (1) the second polypeptide comprises SEQ ID NO: 4 or a polypeptide that is 95% identical to SEQ ID NO: 4 having sucrose phosphate synthase (SPS) activity and the third polypeptide comprises SEQ ID NO: 6 or a polypeptide that is 95% identical to SEQ ID NO: 6 having sucrose phosphate phosphatase (SPP) activity; or (2) the second polypeptide comprises SEQ ID NO: 77 or a polypeptide that is 95% identical to SEQ ID NO: 77 having trehalose phosphate synthase (TPS) activity and the third polypeptide comprises SEQ ID NO: 79 or a polypeptide that is 95% identical to SEQ ID NO: 79 having trehalose phosphate phosphatase (TPP) activity; and
 (iii) a transcriptional termination sequence, 
 wherein the transgenic cyanobacteria accumulates increased levels of the fermentable sugar compared to a cyanobacteria cell not comprising the DNA construct; 
 
 (B) cultivating the transgenic cyanobacteria in or on the photobioreactor; and 
 (C) isolating accumulated fermentable sugar. 
 
     
     
       2. The method of  claim 1 , wherein the fermentable sugar accumulates within the transgenic cyanobacteria. 
     
     
       3. The method of  claim 1 , wherein isolating the accumulated fermentable sugar comprises:
 harvesting at least a portion of the transgenic cyanobacteria from the photobioreactor; and 
 recovering the fermentable sugars from the harvest. 
 
     
     
       4. The method of  claim 1 , wherein the accumulated fermentable sugar is secreted from the transgenic cyanobacteria and isolated from a cultivation media. 
     
     
       5. The method of  claim 1 , wherein isolating the accumulated fermentable sugar comprises isolating the accumulated fermentable sugar from the photobioreactor. 
     
     
       6. The method of  claim 1 , wherein the transgenic cyanobacteria are cultivated to a density of at least about 50 grams of dry biomass per liter equivalent. 
     
     
       7. The method of  claim 1 , wherein the fermentable sugar comprises at least one sugar selected from the group consisting of sucrose and trehalose. 
     
     
       8. The method of  claim 1 , wherein the transgenic cyanobacteria comprises a cyanobacteria selected from the group consisting of  Synechococcus  or  Synechocystis.    
     
     
       9. The method of  claim 1 , further comprising inducing synthesis of the fermentable sugar by the transgenic cyanobacteria. 
     
     
       10. The method of  claim 9 , wherein inducing synthesis of the fermentable sugar comprises exposing the transgenic cyanobacteria to an inducing agent selected from the group consisting of temperature, pH, a metabolite, light, an osmotic agent, a heavy metal, and an antibiotic. 
     
     
       11. The method of  claim 10 , wherein the inducing agent is applied to the photobioreactor by aerosol spray. 
     
     
       12. The method of  claim 9 , wherein inducing synthesis of the fermentable sugar comprises treating the transgenic cyanobacteria with a salt compound. 
     
     
       13. The method of  claim 9 , wherein the salt compound is sodium chloride. 
     
     
       14. The method of  claim 9 , wherein the salt compound is added at a concentration of between about 0.01 mM and 1.5 M or between about 0.2 M and 0.9 M. 
     
     
       15. The method of  claim 1 , wherein,
 the photobioreactor comprises
 (a) a cultivation support, the cultivation support being non-gelatinous, solid, and suitable for providing nutrients and moisture to the cyanobacteria on at least a portion of a surface thereof, wherein said portion of the surface has a topography that allows the cyanobacteria to adhere thereto when said portion of the surface is oriented non-horizontally; and 
 (b) a physical barrier disposed over at least said portion of the surface of the cultivation support; and 
 
 inoculating the photobioreactor with the cyanobacteria comprises inoculating the cultivation support of the photobioreactor. 
 
     
     
       16. The method of  claim 15 , wherein the physical barrier is configured so as to allow inoculation of said portion of the surface of the cultivation support, formation and maintenance of an environment suitable for the cultivation and harvesting of the transgenic cyanobacteria. 
     
     
       17. The method of  claim 15 , further comprising releasably sealing the physical barrier of the photobioreactor after the inoculation of the cultivation support, wherein the cultivation of the transgenic cyanobacteria occurs while the physical barrier is sealed or unsealed. 
     
     
       18. The method of  claim 15 , further comprising at least one of: supplying fluid to the cultivation support; supplying nutrients to the cultivation support; or supplying gas to the cultivation support. 
     
     
       19. The method of  claim 15 , further comprising conveying the photobioreactor to at least one of an inoculation station, a cultivation station, and a harvesting station. 
     
     
       20. A method of producing a fermentable sugar using a photobioreactor, the method comprising:
 (A) inoculating a cultivation support of a photobioreactor with a cyanobacteria transformed with the nucleotide sequence of  claim 1  that produces a fermentable sugar, wherein the photobioreactor comprises 
 (i) the cultivation support, the cultivation support being non-gelatinous, solid, and suitable for providing nutrients and moisture to the photosynthetic microorganisms on at least a portion of a surface thereof, wherein said portion of the surface has a topography that allows the transgenic cyanobacteria to adhere thereto when said portion of the surface is oriented non-horizontally; and 
 (ii) a physical barrier disposed over at least said portion of the surface of the cultivation support; and 
 (B) cultivating the cyanobacteria on the cultivation support; and 
 (C) isolating the fermentable sugar.